RNA polymerase is the principal enzyme of gene expression and a target for genetic regulation. Its basic structure-functional features are highly conserved among all living organisms. The broad goal of this project is the understanding of the molecular mechanism of RNA polymerase function and regulation in relation to structure using the molecular genetic approach. To this end, single amino acid substitutions, insertions and deletions will be generated in the cloned genes rpoB and rpoC which encode the two largest subunits of RNA polymerase. The mutant subunits and their fragments will then be overexpressed from rpo expression plasmids, and the mutant enzyme will be isolated from cells or reconstituted from individual subunits and their fragments in vitro. The malfunctioning of RNA polymerase caused by the mutations will be characterized using an arsenal of in vitro assays which allow to monitor and discriminate between individual steps of RNA polymerase functional cycle. Particular emphasis will be given to the analysis of dominant lethal mutants which disrupt vital basic functions of RNA polymerase. This approach is anticipated to provide molecular details of promoter binding, the catalytic reaction and the mechanism of elongation and termination of RNA. The experiments with deletions and subunit fragments are expected to dissect the two large subunits of RNA polymerase into individual domains. These results will help understanding of the general architecture of RNA polymerase and will become an important resource for direct structural studies. Testing of the mutant polymerase in in vitro assays of regulated transcription will help identify target sites for regulatory factors. Experiments are also proposed on the interaction of RNA polymerase with antibiotics rifampicin and streptolidigin which have direct public health significance.